Prostaglandin and prostamide drug delivery systems and intraocular therapeutic uses thereof

ABSTRACT

Biocompatible, bioerodible implants and microspheres include latanoprost and a biodegradable polymer effective, when placed intraocular (such as into the subtenon space) to treat glaucoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of copending U.S. patentapplication Ser. No. 13/596,904, filed on Aug. 28, 2012, which is adivisional of U.S. patent application Ser. No. 12/259,153, filed on Oct.27, 2008, now abandoned, the entire disclosure of both applications areincorporated herein by reference.

BACKGROUND

The present invention relates to prostaglandin and/or prostamide (aswell as to esters, salts and derivatives thereof) drug delivery systemsfor intraocular use. Prostaglandins are a class of pharmacologicallyactive hormone like substances made in various mammalian tissues, whichare derived from arachidonic acid, and mediate a wide range ofphysiological functions including blood pressure, smooth musclecontraction and inflammation. Examples of prostaglandins areprostaglandin E₁ (alprostadil), prostaglandin E₂ (dinoprostone),latanoprost and travoprost. Latanoprost and travoprost are actuallyprostaglandin prodrugs (i.e. 1-isopropyl esters of a prostaglandin)however, they are referred to as prostaglandins because they act on theprostaglandin F receptor, after being hydrolyzed to the 1-carboxylicacid. A prostamide (also called a prostaglandin-ethanolamide) is aprostaglandin analogue, which is pharmacologically unique from aprostaglandin (i.e. because prostamides act on a different cell receptor[the prostamide receptor] than do prostaglandins), and is a neutrallipid formed a as product of cyclo-oxygenase-2 (“COX-2”) enzymeoxygenation of an endocannabinoid (such as anandamide). Additionally,prostamides do not hydrolyze in-situ to the 1-carboxylic acid. Examplesof prostamides are bimatoprost (the synthetically made ethyl amide of17-phenyl prostaglandin F_(2α)) and prostamide F_(2α). The drug deliverysystems of our invention can be a drug containing implant or implants ora plurality of drug containing microspheres (synonymously“microparticles”). The drug delivery system can be used therapeuticallyto treat an ocular disease or condition.

Glaucoma is a disease of the eye characterized by increased intraocularpressure. Glaucoma can be classified as primary or secondary. Primaryglaucoma in adults (congenital glaucoma) may be either open-angle oracute or chronic angle-closure. Secondary glaucoma results frompre-existing ocular diseases such as uveitis, intraocular tumor or anenlarged cataract.

Certain prostaglandins and their analogs and derivatives, such as thePGF_(2α) derivative (sometimes referred to as prostaglandin F2αanalogue) latanoprost, sold under the trademark Xalatan®, have been usedto treat ocular hypertension and glaucoma. Unfortunately, when topicallyadministered (i.e. as eye drops) latanoprost can have the undesirableside effects of changing the patient's eye color (by increasing irisbrown pigment) and causing hyperemia (eye redness or inflammation).Hence, topical administration of latanoprost can be undesirable for bothcosmetic and patient comfort reasons.

Intraocular prostaglandin and prostamide implant and microspheres aredisclosed by U.S. patent application Ser. Nos. 11/368,845; 11/303,462,and; 10/837,260.

Thus it would be advantageous to provide sustained release latanoprostcontaining implants and microspheres for intraocular (as opposed totopical) therapeutic use to treat glaucoma, with few or no negative sideeffects, such as no or substantially no change to iris pigmentation orhyperemia. Such implants or microspheres by releasing the drug over amultiday period can provide an alternative to daily topicaladministration of latanoprost eyedrops to lower intraocular pressure(“IOP”), thereby treating glaucoma with increased patient compliance(since daily dosing is not required) using a sustained releaseformulation (latanoprost containing microspheres).

SUMMARY

The present invention provides a novel pharmaceutical composition andglaucoma treatment methods. The composition is in the form of an implantor microspheres which advantageously provide for extended release timesof one or more therapeutic agents which is a prostaglandin or aprostamide, such as latanoprost. The implants or the microspheres canrelease the drug over a relatively long period of time, for example, forat least about one week or for example for between about two months andabout six months, after intraocular (i.e. intrascleral) administrationof latanoprost containing implant or microspheres. Such extended releasetimes facilitate obtaining successful treatment results. In addition,administering such implant or microparticles subconjunctivally (i.e.sub-tenon) can reduce the occurrence and/or severity of at least oneside effect, for example, iris color change and/or hyperemia, relativeto administering an identical amount of the latanoprost to the eye inthe form of a topical composition.

An embodiment of our invention is a pharmaceutical composition forintraocular use to treat an ocular condition. The composition cancomprise a plurality of microspheres made of a bioerodible polymer, anda therapeutic agent selected from the group consisting of latanoprost,bimatoprost and travoprost and their salts, esters and derivatives,contained by the microspheres. The microspheres can comprise from about1% to about 99% by weight of the polymer and the polymer can be a PLGAand/or PLA. Additionally, the microspheres can have an average greatestdimension in a range of from about 5 microns to about 1 mm, for examplethe microspheres can have a mean diameter between about 15 microns andabout 55 microns and the therapeutic agent can comprise from about 0.1%to about 90% by weight of the microspheres, such as between about 8 to15 weight % latanoprost.

In another embodiment of our invention the composition can include ahigh viscosity hyaluronic acid and the ocular condition treated can beglaucoma.

A detailed embodiment of our invention is a pharmaceutical compositionfor intraocular use to treat glaucoma comprising a plurality ofmicrospheres made from a PLGA and/or PLA, latanoprost contained by themicrospheres, and a high viscosity hyaluronic acid.

Another embodiment of our invention is a pharmaceutical composition forintraocular use to treat glaucoma, the composition comprising asustained release implant made from a PLGA polymer, a PLA polymer, and aPEG co-solvent, and; latanoprost contained by the implant, wherein theimplant comprises about 30 weight percent latanoprost and the implantcan release the latanoprost over a period of time of at least 20, 30,40, 50, 60, 70 or up to 180 days.

Another embodiment of our invention is a method of treating glaucoma,the method comprising intraocular administration to a patient withglaucoma of a pharmaceutical composition comprising the implant setforth above or a plurality of microspheres made from a PLGA and/or PLA;latanoprost contained by the microspheres, and a high viscosityhyaluronic acid (HA), thereby treating the glaucoma. Preferably, the HAis used with the plurality of microspheres formulation but not with thesingle implant administered. The microspheres can release thelatanoprost for at least about one week after the administration step.The intraocular administration step can be carried out by injection intothe sub-tenon space, such as into the anterior sub-tenon space and thepharmaceutical composition treats glaucoma by reducing baselineintraocular pressure by up to 20%, 30%, 40% or up to 45%.

Within the scope of our invention is a formulation of latanoprostcontaining microspheres which can release therapeutically effectiveamounts of the latanoprost from the microspheres in vivo (i.e. uponintraocular administration of the microspheres) over a period of timefrom about 1 week to about six months, including for a period of time oftwo, three, four, five or six months.

Another embodiment of our invention is a method of treating glaucoma,the method comprising anterior sub-tenon administration to a patientwith glaucoma of a pharmaceutical composition comprising a plurality ofmicrospheres made from a PLGA and/or PLA; latanoprost contained by themicrospheres, wherein the microspheres release the latanoprost for atleast about one week (such as for about two months) after theadministration step, and; a high viscosity hyaluronic acid, therebytreating the glaucoma by reducing baseline intraocular pressure by up toabout 45%.

Additional aspects and advantages of the present invention are set forthin the following description and claims, particularly when considered inconjunction with the accompanying drawings.

DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 is a table showing characteristics of sixteen (MP-2 to MP-17)different batches of latanoprost containing microspheres made by themethod of Example 1, showing the bioerodible polymer(s) used, the amountof latanoprost released form each type of microsphere after one day invitro, the weight percent of latanoprost loaded in the microspheres and,the diameter of the latanoprost containing microspheres made before andafter lyophilization.

FIG. 2 is graph showing in vitro percent cumulative release oflatanoprost from three samples of the FIG. 1 microspheres, over a 15 dayperiod.

FIG. 3 is a graph showing in vivo (in dogs) percent change from baselineIOP over a 28 day period after sub-tenon injection of the FIG. 1 MP-5microspheres in three different formulations (a 50 ul concentration, a100 ul less concentrated formulation, and the 100 ul less concentratedformulation with Captique).

FIG. 4 is a cross sectional view of a diagramatic representation of aportion of a human eye showing (the arrows) the direction of flow ofaqueous humor made by the ciliary body away from the ciliary body.

FIG. 5 is cross sectional view of a diagramatic representation of thesame portion of a human eye shown in FIG. 4 with the arrows now showingthat the flow of aqueous humor away from the ciliary body carriessubstantial amounts of anterior sub-tenon injected away from the site ofinjection into the anterior chamber of the eye.

FIG. 6 is a graph showing in vitro release over a 70 day period oflatanoprost from the implants made in Example 4.

DESCRIPTION

Our invention is based on the discovery of a novel pharmaceuticalcomposition comprising latanoprost containing implants or microspheresand their effective use to treat glaucoma with (as compared to daily,topical Xalatan) reduced iris pigmentation, less hyperemia and manyfewer required drug administrations (i.e. 3-8 times a year injection oflatanoprost containing microspheres according to our invention asopposed to daily Xalatan eyedrops).

With the present latanoprost containing implant or microspheres theamount of the latanoprost released into the eye for a period of timegreater than about one week after the implant or microparticles areplaced in the eye is effective in treating or reducing a symptom of anocular condition, such as ocular hypertension or a retinal degenerationdisease or condition.

DEFINITIONS

The following definitions are used herein.

“About” means plus or minus ten percent of the number, parameter orcharacteristic so qualified.

“Microsphere” and “microparticle” are used synonymously to refer to asmall diameter or dimension (see below) device or element that isstructured, sized, or otherwise configured to be administeredsubconjunctivally (i.e. sub-tenon). Microspheres or microparticlesincludes particles, micro or nanospheres, small fragments,microparticles, nanoparticles, fine powders and the like comprising abiocompatible matrix encapsulating or incorporating a therapeutic agent.Microspheres are generally biocompatible with physiological conditionsof an eye and do not cause significant adverse side effects.Microspheres administered subconjunctivally can be used safely withoutdisrupting vision of the eye. Microspheres have a maximum dimension,such as diameter or length, less than 1 mm. For example, microparticlescan have a maximum dimension less than about 500 μm. Microspheres canalso have a maximum dimension no greater than about 200 μm, or may havea maximum dimension from about 30 μm to about 50 μm, among other sizes.An “implant” is a drug delivery device which is considerably larger thana microsphere, and whereas a plurality (i.e. hundreds or thousands)) ofmicrospheres are administered to treat an ocular condition (such asglaucoma) usually only one to at most six implants are administered forthe same purpose.

“Ocular region” or “ocular site” means any area of the eyeball,including the anterior and posterior segment of the eye, and whichgenerally includes, but is not limited to, any functional (e.g., forvision) or structural tissues found in the eyeball, or tissues orcellular layers that partly or completely line the interior or exteriorof the eyeball. Specific examples of areas of the eyeball in an ocularregion include the anterior chamber, the posterior chamber, the vitreouscavity, the choroid, the suprachoroidal space, the conjunctiva, thesubconjunctival space, the episcleral space, the intracorneal space, theepicorneal space, the sclera, the pars plana, surgically-inducedavascular regions, the macula, and the retina.

“Ocular condition” means a disease, ailment or condition which affectsor involves the eye or one of the parts or regions of the eye. Broadlyspeaking the eye includes the eyeball and the tissues and fluids whichconstitute the eyeball, the periocular muscles (such as the oblique andrectus muscles) and the portion of the optic nerve which is within oradjacent to the eyeball.

An anterior ocular condition is a disease, ailment or condition whichaffects or which involves an anterior (i.e. front of the eye) ocularregion or site, such as a periocular muscle, an eye lid or an eye balltissue or fluid which is located anterior to the posterior wall of thelens capsule or ciliary muscles. Thus, an anterior ocular conditionprimarily affects or involves the conjunctiva, the cornea, the anteriorchamber, the iris, the posterior chamber (behind the retina but in frontof the posterior wall of the lens capsule), the lens or the lens capsuleand blood vessels and nerve which vascularize or innervate an anteriorocular region or site.

Thus, an anterior ocular condition can include a disease, ailment orcondition, such as for example, aphakia; pseudophakia; astigmatism;blepharospasm; cataract; conjunctival diseases; conjunctivitis; cornealdiseases; corneal ulcer; dry eye syndromes; eyelid diseases; lacrimalapparatus diseases; lacrimal duct obstruction; myopia; presbyopia; pupildisorders; refractive disorders and strabismus. Glaucoma can also beconsidered to be an anterior ocular condition because a clinical goal ofglaucoma treatment can be to reduce a hypertension of aqueous fluid inthe anterior chamber of the eye (i.e. reduce intraocular pressure).

A posterior ocular condition is a disease, ailment or condition whichprimarily affects or involves a posterior ocular region or site such aschoroid or sclera (in a position posterior to a plane through theposterior wall of the lens capsule), vitreous, vitreous chamber, retina,optic nerve (i.e. the optic disc), and blood vessels and nerves whichvascularize or innervate a posterior ocular region or site.

Thus, a posterior ocular condition can include a disease, ailment orcondition, such as for example, acute macular neuroretinopathy; Behcet'sdisease; choroidal neovascularization; diabetic uveitis; histoplasmosis;infections, such as fungal or viral-caused infections; maculardegeneration, such as acute macular degeneration, non-exudative agerelated macular degeneration and exudative age related maculardegeneration; edema, such as macular edema, cystoid macular edema anddiabetic macular edema; multifocal choroiditis; ocular trauma whichaffects a posterior ocular site or location; ocular tumors; retinaldisorders, such as central retinal vein occlusion, diabetic retinopathy(including proliferative diabetic retinopathy), proliferativevitreoretinopathy (PVR), retinal arterial occlusive disease, retinaldetachment, uveitic retinal disease; sympathetic opthalmia; VogtKoyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocularcondition caused by or influenced by an ocular laser treatment;posterior ocular conditions caused by or influenced by a photodynamictherapy, photocoagulation, radiation retinopathy, epiretinal membranedisorders, branch retinal vein occlusion, anterior ischemic opticneuropathy, non-retinopathy diabetic retinal dysfunction, retinitispigmentosa, and glaucoma. Glaucoma can be considered a posterior ocularcondition because the therapeutic goal is to prevent the loss of orreduce the occurrence of loss of vision due to damage to or loss ofretinal cells or optic nerve cells (i.e. neuroprotection).

“Biodegradable polymer” means a polymer or polymers which degrade invivo, and wherein erosion of the polymer or polymers over time occursconcurrent with or subsequent to release of the therapeutic agent. Theterms “biodegradable” and “bioerodible” are equivalent and are usedinterchangeably herein. A biodegradable polymer may be a homopolymer, acopolymer, or a polymer comprising more than two different polymericunits. The polymer can be a gel or hydrogel type polymer, PLA or PLGApolymer or mixtures or derivatives thereof.

“Therapeutically effective amount” means level or amount of agent neededto treat an ocular condition, or reduce or prevent ocular injury ordamage without causing significant negative or adverse side effects tothe eye or a region of the eye. In view of the above, a therapeuticallyeffective amount of a therapeutic agent, such as a latanoprost, is anamount that is effective in reducing at least one symptom of an ocularcondition.

We have developed implants and microspheres which can release drug loadsover various time periods. These implants or microspheres, which wheninserted into the subconjunctival (such as a sub-tenon) space of an eyeprovide therapeutic levels of a prostamide or prostaglandin, such aslatanoprost, for extended periods of time (e.g., for about 1 week ormore). The disclosed implants and microspheres are effective in treatingocular conditions, such as ocular conditions associated with elevatedintraocular pressure, and more specifically in reducing at least onesymptom of glaucoma.

Additionally, we have developed novel methods for making implants andmicrospheres. The latanoprost of the present implants and microspheresis preferably from about 1% to 90% by weight of the microspheres. Morepreferably, the latanoprost is from about 5% to about 30% by weight ofthe implant or microspheres. In a preferred embodiment, the latanoprostcomprises about 10% by weight of the microsphere (e.g., 5%-15%). Inanother embodiment, the latanoprost comprises about 40% by weight of themicrospheres.

Suitable polymeric materials or compositions for use in the implant ormicrospheres include those materials which are compatible, that isbiocompatible, with the eye so as to cause no substantial interferencewith the functioning or physiology of the eye. Such materials preferablyare at least partially and more preferably substantially completelybiodegradable or bioerodible.

Examples of useful polymeric materials include, without limitation, suchmaterials derived from and/or including organic esters and organicethers, which when degraded result in physiologically acceptabledegradation products, including the monomers. Also, polymeric materialsderived from and/or including, anhydrides, amides, orthoesters and thelike, by themselves or in combination with other monomers, may also finduse. The polymeric materials may be addition or condensation polymers,advantageously condensation polymers. The polymeric materials may becross-linked or non-cross-linked, for example not more than lightlycross-linked, such as less than about 5%, or less than about 1% of thepolymeric material being cross-linked. For the most part, besides carbonand hydrogen, the polymers will include at least one of oxygen andnitrogen, advantageously oxygen. The oxygen may be present as oxy, e.g.hydroxy or ether, carbonyl, e.g. non-oxo-carbonyl, such as carboxylicacid ester, and the like. The nitrogen may be present as amide, cyanoand amino. The polymers set forth in Heller, Biodegradable Polymers inControlled Drug Delivery, In: CRC Critical Reviews in Therapeutic DrugCarrier Systems, Vol. 1, CRC Press, Boca Raton, Fla. 1987, pp 39-90,which describes encapsulation for controlled drug delivery, may find usein the present microspheres.

Of additional interest are polymers of hydroxyaliphatic carboxylicacids, either homopolymers or copolymers, and polysaccharides.Polyesters of interest include polymers of D-lactic acid, L-lactic acid,racemic lactic acid, glycolic acid, polycaprolactone, and combinationsthereof. Generally, by employing the L-lactate or D-lactate, a slowlyeroding polymer or polymeric material is achieved, while erosion issubstantially enhanced with the lactate racemate.

Among the useful polysaccharides are, without limitation, calciumalginate, and functionalized celluloses, particularlycarboxymethylcellulose esters characterized by being water insoluble, amolecular weight of about 5 kD to 500 kD, for example.

Other polymers of interest include, without limitation, polyvinylalcohol, polyesters, polyethers and combinations thereof which arebiocompatible and may be biodegradable and/or bioerodible.

Some preferred characteristics of the polymers or polymeric materialsfor use in the present invention may include biocompatibility,compatibility with the selected therapeutic agent, ease of use of thepolymer in making the drug delivery systems of the present invention, ahalf-life in the physiological environment of at least about 6 hours,preferably greater than about one day, and water insolubility.

The biodegradable polymeric materials which are included to form thematrix are desirably subject to enzymatic or hydrolytic instability.Water soluble polymers may be cross-linked with hydrolytic orbiodegradable unstable cross-links to provide useful water insolublepolymers. The degree of stability can be varied widely, depending uponthe choice of monomer, whether a homopolymer or copolymer is employed,employing mixtures of polymers, and whether the polymer includesterminal acid groups.

Equally important to controlling the biodegradation of the polymer andhence the extended release profile of the implant is the relativeaverage molecular weight of the polymeric composition employed in themicrospheres. Different molecular weights of the same or differentpolymeric compositions may be included in the microspheres to modulatethe release profile. For latanoprost implants, the relative averagemolecular weight of the polymer will preferably range from about 4 toabout 25 kD, more preferably from about 5 to about 20 kD, and mostpreferably from about 5 to about 15 kD.

In some implants and microspheres, copolymers of glycolic acid andlactic acid are used, where the rate of biodegradation is controlled bythe ratio of glycolic acid to lactic acid. The most rapidly degradedcopolymer has roughly equal amounts of glycolic acid and lactic acid.Homopolymers, or copolymers having ratios other than equal, are moreresistant to degradation. The ratio of glycolic acid to lactic acid willalso affect the brittleness of the microspheres. The percentage ofpolylactic acid in the polylactic acid polyglycolic acid (PLGA)copolymer can be 0-100%, preferably about 15-85%, more preferably about35-65%. In some implants, a 50/50 PLGA copolymer is used.

The biodegradable polymer matrix of the subconjunctival implants andmicrospheres may comprise a mixture of two or more biodegradablepolymers. For example, the implants and microspheres may comprise amixture of a first biodegradable polymer and a different secondbiodegradable polymer. One or more of the biodegradable polymers mayhave terminal acid groups.

Release of a drug from an erodible polymer is the consequence of severalmechanisms or combinations of mechanisms. Some of these mechanismsinclude desorption from the implant or microsphere's surface,dissolution, diffusion through porous channels of the hydrated polymerand erosion. Erosion can be bulk or surface or a combination of both. Asdiscussed herein, the matrix of the microspheres may release drug at arate effective to sustain release of an amount of the prostamidecomponent for more than one week after implantation into an eye. Incertain microspheres, therapeutic amounts of latanoprost are releasedfor no more than about 3-30 days after administration to thesubconjunctival space. For example, a microsphere may compriselatanoprost, and the matrix of the microsphere degrades at a rateeffective to sustain release of a therapeutically effective amount oflatanoprost for about one month after being placed under theconjunctiva. As another example, the microspheres may compriselatanoprost, and the matrix releases drug at a rate effective to sustainrelease of a therapeutically effective amount of latanoprost for morethan thirty days, such as for about six months.

One example of the biodegradable implant or microsphere compriseslatanoprost associated with a biodegradable polymer matrix, whichcomprises a mixture of different biodegradable polymers. At least one ofthe biodegradable polymers is a polylactide having a molecular weight ofabout 63.3 kD. A second biodegradable polymer is a polylactide having amolecular weight of about 14 kD. Such a mixture is effective insustaining release of a therapeutically effective amount of thelatanoprost for a time period greater than about one month from the timethe microspheres are placed administered under the conjuctiva.

Another example of a biodegradable implant or microsphere compriseslatanoprost associated with a biodegradable polymer matrix, whichcomprises a mixture of different biodegradable polymers, eachbiodegradable polymer having an inherent viscosity from about 0.16 dl/gto about 1.0 dl/g. For example, one of the biodegradable polymers mayhave an inherent viscosity of about 0.3 dl/g. A second biodegradablepolymer may have an inherent viscosity of about 1.0 dl/g. Additionalmicrospheres may comprise biodegradable polymers that have an inherentviscosity between about 0.2 dl/g and 0.5 dl/g. The inherent viscositiesidentified above may be determined in 0.1% chloroform at 25° C.

The release of the latanoprost from an implant or microspheres into thesubconjuctiva may include an initial burst of release followed by agradual increase in the amount of the latanoprost released, or therelease may include an initial delay in release of the prostamidecomponent followed by an increase in release. When the microspheres aresubstantially completely degraded, the percent of the latanoprost thathas been released is about one hundred. The implants and microspheresdisclosed herein do not completely release, or release about 100% of thelatanoprost, until after about one week of being placed in an eye.

It may be desirable to provide a relatively constant rate of release ofthe latanoprost from the microspheres over the life of the implanted orinjected microspheres. For example, it may be desirable for thelatanoprost to be released in amounts from about 0.01 μg to about 2 μgper day for the life of the microspheres. However, the release rate maychange to either increase or decrease depending on the formulation ofthe biodegradable polymer matrix. In addition, the release profile ofthe prostamide component may include one or more linear portions and/orone or more non-linear portions. Preferably, the release rate is greaterthan zero once the microspheres has begun to degrade or erode.

The implants and microspheres can be monolithic, i.e. having the activeagent or agents homogenously distributed through the polymeric matrix,or encapsulated, where a reservoir of active agent is encapsulated bythe polymeric matrix. Due to ease of manufacture, monolithic implantsare usually preferred over encapsulated forms. However, the greatercontrol afforded by the encapsulated microspheres may be of benefit insome circumstances, where the therapeutic level of the drug falls withina narrow window. In addition, the therapeutic component, including thelatanoprost component, may be distributed in a non-homogenous pattern inthe matrix. For example, the microspheres may include a portion that hasa greater concentration of the latanoprost relative to a second portionof the microspheres.

The implants and microspheres disclosed herein may have a size ofbetween about 5 μm and about 1 mm, or between about 10 μm and about 0.8mm for administration with a needle. For needle-injected microspheres,the microsphere may have any appropriate dimensions so long as thelongest dimension of the microsphere permits the microsphere to movethrough a needle. This is generally not a problem in the administrationof microspheres. The subconjunctival space in humans is able toaccommodate relatively large volumes of microspheres, for example, about100 μl, or about 150 μl, or about 50-200 μl or more.

The total weight of implant or microsphere in a single dosage an optimalamount, depending on the volume of the subconjunctival space and theactivity or solubility of the active agent. Most often, the dose isusually about 0.1 mg to about 200 mg of implant or microspheres perdose. For example, a single subconjunctival injection may contain about1 mg, 3 mg, or about 5 mg, or about 8 mg, or about 10 mg, or about 100mg or about 150 mg, or about 175 mg, or about 200 mg of microspheres,including the incorporated therapeutic component. For non-humanindividuals, the dimensions and total weight of the implant ormicrosphere(s) may be larger or smaller, depending on the type ofindividual.

The dosage of the therapeutic component (i.e. latanoprost) in theimplant or microspheres is generally in the range from about 0.001% toabout 100 mg per eye per dose, but also can vary from this dependingupon the activity of the agent and its solubility.

Thus, implants or microspheres can be prepared where the center may beof one material and the surface may have one or more layers of the sameor a different composition, where the layers may be cross-linked, or ofa different molecular weight, different density or porosity, or thelike. For example, where it is desirable to quickly release an initialbolus of drug, the center of the microsphere may be a polylactate coatedwith a polylactate-polyglycolate copolymer, so as to enhance the rate ofinitial degradation. Alternatively, the center may be polyvinyl alcoholcoated with polylactate, so that upon degradation of the polylactateexterior the center would dissolve and be rapidly washed out of the eye.

The implant or microspheres may be of any particulate geometry includingmicro and nanospheres, micro and nanoparticles, spheres, powders,fragments and the like. The upper limit for the microsphere size will bedetermined by factors such as toleration for the implant, sizelimitations on insertion, desired rate of release, ease of handling,etc. Spheres may be in the range of about 0.5 μm to 4 mm in diameter,with comparable volumes for other shaped particles.

The size and form of the implant or microspheres can also be used tocontrol the rate of release, period of treatment, and drug concentrationat the site of implantation. Larger microspheres will deliver aproportionately larger dose, but depending on the surface to mass ratio,may have a slower release rate. The particular size and geometry of theimplant or microspheres are chosen to suit the activity of the activeagent and the location of its target tissue.

The proportions of the latanoprost, polymer, and any other modifiers maybe empirically determined by formulating several microsphere batcheswith varying average proportions. A USP approved method for dissolutionor release test can be used to measure the rate of release (USP 23; NF18 (1995) pp. 1790-1798). For example, using the infinite sink method, aweighed sample of the microspheres is added to a measured volume of asolution containing 0.9% NaCl in water, where the solution volume willbe such that the drug concentration is after release is less than 5% ofsaturation. The mixture is maintained at 37° C. and stirred slowly tomaintain the microspheres in suspension. The appearance of the dissolveddrug as a function of time may be followed by various methods known inthe art, such as spectrophotometrically, HPLC, mass spectroscopy, etc.until the absorbance becomes constant or until greater than 90% of thedrug has been released.

In addition to the latanoprost included in the implants and microspheresdisclosed herein, the microsphere may also include one or moreadditional ophthalmically acceptable therapeutic agents. For example,the microspheres may include one or more antihistamines, one or moreantibiotics, one or more beta blockers, one or more steroids, one ormore antineoplastic agents, one or more immunosuppressive agents, one ormore antiviral agents, one or more antioxidant agents, and mixturesthereof. Alternatively, a single injection of implant or microspherescan include two or more microsphere batches each containing a differenttherapeutic component or components. Such a mixture of differentimplants and microspheres in included within the present invention.

Additional pharmacologic or therapeutic agents which may find use in thepresent systems, include, without limitation, those disclosed in U.S.Pat. No. 4,474,451, columns 4-6 and 4,327,725, columns 7-8.

Examples of antihistamines include, and are not limited to, loradatine,hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine,cyproheptadine, terfenadine, clemastine, triprolidine, carbinoxamine,diphenylpyraline, phenindamine, azatadine, tripelennamine,dexchlorpheniramine, dexbrompheniramine, methdilazine, and trimprazinedoxylamine, pheniramine, pyrilamine, chiorcyclizine, thonzylamine, andderivatives thereof.

Examples of antibiotics include without limitation, cefazolin,cephradine, cefaclor, cephapirin, ceftizoxime, cefoperazone, cefotetan,cefutoxime, cefotaxime, cefadroxil, ceftazidime, cephalexin,cephalothin, cefamandole, cefoxitin, cefonicid, ceforanide, ceftriaxone,cefadroxil, cephradine, cefuroxime, ampicillin, amoxicillin,cyclacillin, ampicillin, penicillin G, penicillin V potassium,piperacillin, oxacillin, bacampicillin, cloxacillin, ticarcillin,azlocillin, carbenicillin, methicillin, nafcillin, erythromycin,tetracycline, doxycycline, minocycline, aztreonam, chloramphenicol,ciprofloxacin hydrochloride, clindamycin, metronidazole, gentamicin,lincomycin, tobramycin, vancomycin, polymyxin B sulfate, colistimethate,colistin, azithromycin, augmentin, sulfamethoxazole, trimethoprim, andderivatives thereof.

Examples of beta blockers include acebutolol, atenolol, labetalol,metoprolol, propranolol, timolol, and derivatives thereof.

Examples of steroids include corticosteroids, such as cortisone,prednisolone, flurometholone, dexamethasone, medrysone, loteprednol,fluazacort, hydrocortisone, prednisone, betamethasone, prednisone,methylprednisolone, riamcinolone hexacatonide, paramethasone acetate,diflorasone, fluocinonide, fluocinolone, triamcinolone, derivativesthereof, and mixtures thereof.

Examples of antineoplastic agents include adriamycin, cyclophosphamide,actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin,mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU),methyl-CCNU, cisplatin, etoposide, interferons, camptothecin andderivatives thereof, phenesterine, taxol and derivatives thereof,taxotere and derivatives thereof, vinblastine, vincristine, tamoxifen,etoposide, piposulfan, cyclophosphamide, and flutamide, and derivativesthereof.

Examples of immunosuppressive agents include cyclosporine, azathioprine,tacrolimus, and derivatives thereof.

Examples of antiviral agents include interferon gamma, zidovudine,amantadine hydrochloride, ribavirin, acyclovir, valciclovir,dideoxycytidine, phosphonoformic acid, ganciclovir, and derivativesthereof.

Examples of antioxidant agents include ascorbate, alpha-tocopherol,mannitol, reduced glutathione, various carotenoids, cysteine, uric acid,taurine, tyrosine, superoxide dismutase, lutein, zeaxanthin,cryotpxanthin, astazanthin, lycopene, N-acetyl-cysteine, carnosine,gamma-glutamylcysteine, quercitin, lactoferrin, dihydrolipoic acid,citrate, Ginkgo Biloba extract, tea catechins, bilberry extract,vitamins E or esters of vitamin E, retinyl palmitate, and derivativesthereof.

Other therapeutic agents include squalamine, carbonic anhydraseinhibitors, alpha-2 adrenergic receptor agonists, antiparasitics,antifungals, and derivatives thereof.

The amount of active agent or agents employed in the microspheres,individually or in combination, will vary widely depending on theeffective dosage required and the desired rate of release from themicrospheres. Usually the agent will be at least about 1, more usuallyat least about 10 weight percent of the microsphere, and usually notmore than about 80, more usually not more than about 40 weight percentof the microspheres.

Some of the present implants and microspheres can comprise a combinationof two or more different latanoprost derivatives. One microsphere ordosage of microspheres may comprise a combination of bimatoprost andlatanoprost. Another microsphere or dosage of microspheres may comprisea combination of bimatoprost and travoprost.

As discussed herein, the present implants and microspheres may compriseadditional therapeutic agents. For example, one implant or microspheresdosage can comprise a combination of latanoprost and a beta-adrenergicreceptor antagonist. More specifically, the microsphere or dosage ofmicrospheres may comprise a combination of latanoprost and Timolol®. Or,a microsphere or dosage of microspheres may comprise a combination oflatanoprost and a carbonic anyhdrase inhibitor. For example, themicrosphere or dosage of microspheres may comprise a combination oflatanoprost and dorzolamide (Trusopt®).

In addition to the therapeutic component, the implants and microspheresdisclosed herein may include or may be provided in compositions thatinclude effective amounts of buffering agents, preservatives and thelike. Suitable water soluble buffering agents include, withoutlimitation, alkali and alkaline earth carbonates, phosphates,bicarbonates, citrates, borates, acetates, succinates and the like, suchas sodium phosphate, citrate, borate, acetate, bicarbonate, carbonateand the like. These agents advantageously present in amounts sufficientto maintain a pH of the system of between about 2 to about 9 and morepreferably about 4 to about 8. As such the buffering agent may be asmuch as about 5% by weight of the total implant. Suitable water solublepreservatives include sodium bisulfite, sodium bisulfate, sodiumthiosulfate, ascorbate, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, parabens, methylparaben, polyvinyl alcohol,benzyl alcohol, phenylethanol and the like and mixtures thereof. Theseagents may be present in amounts of from about 0.001% to about 5% byweight and preferably about 0.01% to about 2% by weight. In at least oneof the present microspheres, a benzylalkonium chloride preservative isprovided in the implant, such as when the latanoprost consistsessentially of bimatoprost.

In some situations mixtures of implants and microspheres may be utilizedemploying the same or different pharmacological agents. In this way, acocktail of release profiles, giving a biphasic or triphasic releasewith a single administration is achieved, where the pattern of releasemay be greatly varied.

Additionally, release modulators such as those described in U.S. Pat.No. 5,869,079 may be included in the implants or microspheres. Theamount of release modulator employed will be dependent on the desiredrelease profile, the activity of the modulator, and on the releaseprofile of the latanoprost in the absence of modulator. Electrolytessuch as sodium chloride and potassium chloride may also be included inthe microspheres. Where the buffering agent or enhancer is hydrophilic,it may also act as a release accelerator. Hydrophilic additives act toincrease the release rates through faster dissolution of the materialsurrounding the drug in the microspheres, which increases the surfacearea of the drug exposed, thereby increasing the rate of drugbioerosion. Similarly, a hydrophobic buffering agent or enhancerdissolves more slowly, slowing the exposure of drug, and thereby slowingthe rate of drug bioerosion.

In certain microspheres, the combination of latanoprost and abiodegradable polymer matrix is released or delivered an amount oflatanoprost between about 0.1 mg to about 0.5 mg for about 3-6 monthsafter implantation or injection into the eye.

Various techniques may be employed to produce the implants and/ormicrospheres described herein. Useful techniques include, but are notnecessarily limited to, self-emulsification methods, super criticalfluid methods, solvent evaporation methods, phase separation methods,spray drying methods, grinding methods, interfacial methods, moldingmethods, injection molding methods, combinations thereof and the like.

As discussed herein, the polymeric component recited in the presentmethod may comprise a biodegradable polymer or biodegradable copolymer.In at least one embodiment, the polymeric component comprises a poly(lactide-co-glycolide) PLGA copolymer. In a further embodiment, the PLGAcopolymer has a lactide/glycolide ratio of 75/25. In a still furtherembodiment, the PLGA copolymer has at least one of a molecular weight ofabout 63 kilodaltons and an inherent viscosity of about 0.6 dL/g.

The present methods may also comprise a step of forming a firstcomposition which comprises a latanoprost component, a polymericcomponent, and an organic solvent, and a step of forming a secondoil-containing composition, and mixing the first composition and thesecond oil-containing composition.

In addition, the present population of microparticles may have a maximumparticle diameter less than about 200 μm. In certain embodiments, thepopulation of microparticles has an average or mean particle diameterless than about 50 μm. In further embodiments, the population ofmicroparticles has a mean particle diameter from about 30 μm to about 50μm.

The present implants and microparticles are structured or configured torelease the latanoprost for extended periods of time at controlledrates. In some embodiments, the latanoprost is released at asubstantially linear rate (e.g., a single rate) over the life of themicroparticles (e.g., until the microparticles fully degrade). Otherembodiments are capable of releasing the latanoprost at multiple ratesor different rates over the life of the microparticles. The rate atwhich the microparticles degrade can vary, as discussed herein, andtherefore, the present microparticles can release the latanoprost fordifferent periods of time depending on the particular configuration andmaterials of the microparticles. In at least one embodiment, amicroparticle can release about 1% of the latanoprost in themicroparticles per day. In a further embodiment, the microparticles mayhave a release rate of about 0.7% per day when measured in vitro. Thus,over a period of about 40 days, about 30% of the latanoprost may havebeen released.

As discussed herein, the amount of the latanoprost present in theimplants and microspheres can vary. In certain embodiments, about 10 wt% of the microspheres is the latanoprost. In further embodiments, thelatanoprost constitutes about 5 wt % of the microspheres.

The microspheres, including the population of microspheres, of thepresent invention may be inserted into the subconjunctival (i.e.sub-tenon) space of an eye by a variety of methods. The method ofplacement may influence the therapeutic component or drug releasekinetics. A preferred means of administration of the microspheres of thepresent invention is by subconjunctival injection. The location of thesite of injection of the microspheres may influence the concentrationgradients of therapeutic component or drug surrounding the element, andthus influence the delivery rate to a given tissue of the eye. Forexample, an injection into the conjunctiva toward the posterior of theeye will direct drug more efficiently to the tissues of the posteriorsegment, while a site of injection closer to the anterior of the eye(but avoiding the cornea) may direct drug more efficiently to theanterior segment.

Microparticles may be administered to patients by administering anophthalmically acceptable composition which comprises the microparticlesto the patient. For example, microparticles may be provided in a liquidcomposition, a suspension, an emulsion, and the like, and administeredby injection or implantation into the subconjunctival space of the eye.

The present implants or microparticles are configured to release anamount of latanoprost effective to treat an ocular condition, such as byreducing at least one symptom of the ocular condition. Morespecifically, the microparticles may be used in a method to treatglaucoma, such as open angle glaucoma, ocular hypertension, chronicangle-closure glaucoma, with patent iridotomy, psuedoexfoliativeglaucoma, and pigmentary glaucoma. By injecting the latanoprostcomponent-containing microspheres into the subconjunctival space of aneye, it is believed that the latanoprost is effective to enhance aqueoushumor flow thereby reducing intraocular pressure. Additionally, thepresent inventors have shown that subconjunctival delivery ofmicrospheres containing latanoprost is able to provide quite highconcentrations of the therapeutic agent to the retina of the eye.

The latanoprost containing implants and microspheres disclosed hereinmay also be configured to release the latanoprost with or withoutadditional agents, as described above, which to prevent or treatdiseases or conditions, such as the following: maculopathies/retinaldegeneration: macular degeneration, including age related maculardegeneration (ARMD), such as non-exudative age related maculardegeneration and exudative age related macular degeneration, choroidalneovascularization, retinopathy, including diabetic retinopathy, acuteand chronic macular neuroretinopathy, central serous chorioretinopathy,and macular edema, including cystoid macular edema, and diabetic macularedema. Uveitis/retinitis/choroiditis: acute multifocal placoid pigmentepitheliopathy, Behcet's disease, birdshot retinochoroidopathy,infectious (syphilis, lyme, tuberculosis, toxoplasmosis), uveitis,including intermediate uveitis (pars planitis) and anterior uveitis,multifocal choroiditis, multiple evanescent white dot syndrome (MEWDS),ocular sarcoidosis, posterior scleritis, serpignous choroiditis,subretinal fibrosis, uveitis syndrome, and Vogt-Koyanagi-Haradasyndrome. Vascular diseases/exudative diseases: retinal arterialocclusive disease, central retinal vein occlusion, disseminatedintravascular coagulopathy, branch retinal vein occlusion, hypertensivefundus changes, ocular ischemic syndrome, retinal arterialmicroaneurysms, Coat's disease, parafoveal telangiectasis, hemi-retinalvein occlusion, papillophlebitis, central retinal artery occlusion,branch retinal artery occlusion, carotid artery disease (CAD), frostedbranch angitis, sickle cell retinopathy and other hemoglobinopathies,angioid streaks, familial exudative vitreoretinopathy, Eales disease.Traumatic/surgical: sympathetic ophthalmia, uveitic retinal disease,retinal detachment, trauma, laser, PDT, photocoagulation, hypoperfusionduring surgery, radiation retinopathy, bone marrow transplantretinopathy. Proliferative disorders: proliferative vitreal retinopathyand epiretinal membranes, proliferative diabetic retinopathy. Infectiousdisorders: ocular histoplasmosis, ocular toxocariasis, presumed ocularhistoplasmosis syndrome (POHS), endophthalmitis, toxoplasmosis, retinaldiseases associated with HIV infection, choroidal disease associatedwith HIV infection, uveitic disease associated with HIV Infection, viralretinitis, acute retinal necrosis, progressive outer retinal necrosis,fungal retinal diseases, ocular syphilis, ocular tuberculosis, diffuseunilateral subacute neuroretinitis, and myiasis. Genetic disorders:retinitis pigmentosa, systemic disorders with associated retinaldystrophies, congenital stationary night blindness, cone dystrophies,Stargardt's disease and fundus flavimaculatus, Bests disease, patterndystrophy of the retinal pigmented epithelium, X-linked retinoschisis,Sorsby's fundus dystrophy, benign concentric maculopathy, Bietti'scrystalline dystrophy, pseudoxanthoma elasticum. Retinal tears/holes:retinal detachment, macular hole, giant retinal tear. Tumors: retinaldisease associated with tumors, congenital hypertrophy of the RPE,posterior uveal melanoma, choroidal hemangioma, choroidal osteoma,choroidal metastasis, combined hamartoma of the retina and retinalpigmented epithelium, retinoblastoma, vasoproliferative tumors of theocular fundus, retinal astrocytoma, intraocular lymphoid tumors.Miscellaneous: punctate inner choroidopathy, acute posterior multifocalplacoid pigment epitheliopathy, myopic retinal degeneration, acuteretinal pigment epithelitis and the like.

In one embodiment, an implant, such as the microspheres disclosedherein, is administered to a subconjunctival space of an eye. In atleast one embodiment, a method of reducing intraocular pressure in aneye of a patient comprises administering a microsphere containinglatanoprost, as disclosed herein, to a patient by subconjuctivalinjection. A syringe apparatus including an appropriately sized needle,for example, a 22 gauge needle, a 27 gauge needle or a 30 gauge needle,can be effectively used to inject the composition with into thesubconjunctival space of an eye of a human or animal. Frequent repeatinjections are often not necessary due to the extended release of thelatanoprost from the microspheres.

In addition, for dual therapy approaches to treating an ocularcondition, the method may include one or more additional steps ofadministering additional therapeutic agents to the eye, such as bytopically administering compositions containing timolol, dorzolamide,and iatoprost, among others.

In another aspect of the invention, kits and packages for treating anocular condition of the eye are provided, comprising: a) a containercomprising an extended release microsphere formulation comprising atherapeutic component including latanoprost, and a drug releasesustaining component; and b) instructions for use. Instructions mayinclude steps of how to handle the microspheres, how to insert themicrospheres into an ocular region, and what to expect from using themicrospheres.

In certain implants, the microspheres preparation comprises atherapeutic component which consists essentially of latanoprost, saltsthereof, and mixtures thereof, and a biodegradable polymer matrix. Thebiodegradable polymer matrix may consist essentially of PLA, PLGA, or acombination thereof. When placed in the eye, the preparation releasesabout 40% to about 60% of the latanoprost to provide a loading dose ofthe latanoprost within about one day after subconjunctivaladministration. Subsequently, the microspheres release about 1% to about2% of the latanoprost per day to provide a sustained therapeutic effect.Such implant or microsphere preparations can be effective in reducingand maintaining a reduced intraocular pressure, such as below about 15mm Hg for several months, and potentially for one or two years.

Other microspheres disclosed herein may be configured such that theamount of the latanoprost that is released from the microspheres withintwo days of subconjunctival injection is less than about 95% of thetotal amount of the latanoprost in the microsphere. In certainformulations, 95% of the latanoprost is not released until after aboutone week of injection. In certain microsphere formulations, about 50% ofthe latanoprost is released within about one day of placement in theeye, and about 2% is released for about 1 month after being placed inthe eye. In other microspheres, about 50% of the latanoprost is releasedwithin about one day of subconjunctival administration, and about 1% isreleased for about 2 months after such administration.

In an alternative embodiment, the present invention may comprise theinstallation of a hollow depot device, made from a biocompatible,preferably non-biodegradable, polymer in connection with and preferablypenetrating the conjunctiva of an eye. Such a device is described, forexample, in US Patent Publication 20050112175, hereby incorporated byreference herein. The depot is preferably refillable with microspherescomprising an ophthalmically active therapeutic component, such as anophthalmically active latanoprost.

A pharmaceutical composition (such as an implant or microspheres) withinthe scope of our invention can be formulated with a high viscosity,polymeric gel to reduce dispersion of the composition upon intraocularinjection. Preferably, the gel has a high shear characteristic, meaningthat the gel can be injected into an intraocular site through a 25-30gauge needle, and more preferably through a 27-30 gauge needle. Asuitable gel for this purpose can be a hydrogel or a colloidal gelformed as a dispersion in water or other aqueous medium. Examples ofsuitable gels include synthetic polymers such as polyhydroxy ethylmethacrylate, and chemically or physically crosslinked polyvinylalcohol, polyacrylamide, poly(N-vinyl pyrolidone), polyethylene oxide,and hydrolysed polyacrylonitrile. Examples of suitable hydrogels whichare organic polymers include covalent or ionically crosslinkedpolysaccharide-based hydrogels such as the polyvalent metal salts ofalginate, pectin, carboxymethyl cellulose, heparin, hyaluronate (i.e.polymeric hyaluronic acid) and hydrogels from chitin, chitosan,pullulan, gellan, xanthan and hydroxypropylmethylcellulose. Commerciallyavailable dermal fillers (such as Hylafrom®, Restylane®, Sculptura™ andRadiesse) can be used as the high viscosity gel in embodiments of ourpharmaceutical composition.

Hyaluronic acid (“HA”) is a polysaccharide made by various body tissues.U.S. Pat. No. 5,166,331 discusses purification of different fractions ofhyaluronic acid for use as a substitute for intraocular fluids and as atopical ophthalmic drug carrier. Other U.S. patent applications whichdiscuss ocular uses of hyaluronic acid include serial numbers11/859,627; 11/952,927; 10/966,764; 11/741,366; and 11/039,192 Thepharmaceutical compositions within the scope of our invention preferablycomprise a high viscosity hyaluronic acid with an average molecularweight between about 1 and 4 million Daltons, and more preferably withan average molecular weight between about 2 and 3 million Daltons, andmost preferably with an average molecular weight of about (±10%) 2million Daltons.

Dry uncrosslinked HA material comprises fibers or powder of commerciallyavailable HA, for example, fibers or powder of sodium hyaluronate(NaHA). The HA may be bacterial-sourced sodium hyaluronate, animalderived sodium hyaluronate or a combination thereof. In someembodiments, the dry HA material is a combination of raw materialsincluding HA and at least one other polysaccharide, for example,glycosaminoglycan (GAG).

In our invention the HA used comprises or consists of high molecularweight HA. That is, nearly 100% of the HA material in the presentcompositions is a high molecular weight HA. High molecular weight HAmeans HA with a molecular weight of at least about 1.0 million Daltons(mw≧10⁶ Da) to about 4.0 million Da (mw≦4×10⁶ Da). For example, the highmolecular weight HA in the present compositions may have a molecularweight of about 2.0 million Da (mw 2×10⁶ Da). In another example, thehigh molecular weight HA may have a molecular weight of about 2.8million Da (mw 2.8×10⁶ Da).

In an embodiment of our invention, dry or raw HA material (in thisspecific example, NaHA) having a desired high/low molecular weight ratiois cleaned and purified. These steps generally involved hydrating thedry HA fibers or powder in the desired high/low molecular weight ratio,for example, using pure water, and filtering the material to removelarge foreign matters and/or other impurities. The filtered, hydratedmaterial is then dried and purified. The high and low molecular weightNaHA may be cleaned and purified separately, or may be mixed together,for example, in the desired ratio, just prior to crosslinking.

At this stage in the process, the pure, dried NaHA fibers are hydratedin an alkaline solution to produce an uncrosslinked NaHA alkaline gel.Any suitable alkaline solution may be used to hydrate the NaHA in thisstep, for example, but not limited to an aqueous solution containingNaOH. The resulting alkaline gel will have a pH above 7.5, for example,a pH above 8, for example, a pH above 9, for example, a pH above 10, forexample, a pH above 12, for example, a pH above 13.

In this specific example, the next step in the manufacturing processcomprises the step of crosslinking the hydrated, alkaline NaHA gel witha suitable crosslinking agent, for example, BDDE.

The step of crosslinking may be carried out using means known to thoseof skill in the art. Those skilled in the art appreciate how to optimizethe conditions of crosslinking according to the nature of the HA, andhow to carry out the crosslinking to an optimized degree.

In some embodiments of the present invention, the degree of crosslinkingis at least about 2% to about 20%, for example, is about 4% to about12%, wherein the degree of crosslinking is defined as the percent weightratio of the crosslinking agent to HA-monomeric units in thecomposition.

The hydrated crosslinked, HA gel may be neutralized by adding an aqueoussolution containing HCl. The gel is then swelled in a phosphate bufferedsaline solution for a sufficient time and at a low temperature.

In certain embodiments, the resulting swollen gel (HA) is a cohesive gelhaving substantially no visible distinct particles, for example,substantially no visibly distinct particles when viewed with the nakedeye. In some embodiments, the gel has substantially no visibly distinctparticles under a magnification of less than 35×.

The gel ((HA) is now purified by conventional means for example,dialysis or alcohol precipitation, to recover the crosslinked material,to stabilize the pH of the material and remove any unreactedcrosslinking agent. Additional water or slightly alkaline aqueoussolution can be added to bring the concentration of the NaHA in thecomposition to a desired concentration. In some embodiments, theconcentration of NaHA in the composition is in a range between about 10mg/ml to about 30 mg/ml.

EXAMPLES

The following examples set forth specific, non-limiting embodiments ofour invention

Example 1 Method for Making Latanoprost Microspheres

Latanoprost containing microspheres were made by dissolving 20 mg oflatanoprost and 100 mg polymer (Resomer 203H) in 0.8 ml ethyl acetate. Aminimum amount of dichloromethane was added to complete dissolution.Then added to this solution was 40 ml 1% polyvinyl acetate in water viaa micro-pipette while shearing the mixture at 3000 rpm for 5 minuteswith a Silverson homogenizer. When the polymer solution was added to thewater, the pipette tip was submerged under the water surface and addeddropwise.

After shearing, a milky white emulsion was formed, and it was mildlyagitated in a hood for 3-5 hrs to allow solvent evaporation. Thissuspension was filtered through a 106 um sieves, and particle size wasmeasured. The suspension was then centrifuged at 2000 rpm for 15 min toremove supernatant, followed by adding 10 mL distilled water toreconstitute the microspheres. Finally the microspheres were lyophilizedfollowed by drug content assay, and in vitro release assay. Typicalmicrosphere diameters were about 35 um, with 13% latanoprost loading.

Samples of the microspheres were formulated with a high viscosityhyaluronic acid. Thus, 10 mg microspheres were mixed with 100 uLCaptique gel and another 10 mg microsphere sample was mixed with 50 uLJ18 gel.

FIG. 1 provides information regarding sixteen batches of latanoprostmicrospheres made using various known bioerodible polymers, such asR203H and RG502H. First day in vitro release (in PBS medium with 0.1%triton 100) rates varied from 3% to 60% of the latanoprost, andmicrosphere mean diameter (“PS”, i.e. particle size in FIG. 1) wasbetween 18 and 52 microns. In FIG. 1 “d10” is the mean particle sizepossessed by 10% or less of the total number of microspheres made inthat batch, while “d90” is the mean particle size possessed by 90% orless of the total number of microspheres made in that batch. FIG. 2shows in vitro release of latanoprost from selected microsphereformulations over a 15 day period.

Example 2 In Vivo Use of Latanoprost Microspheres to Lower IntraocularPressure

Experiments were carried out in which the latanoprost containingmicrospheres of Example 1 were administered by subtenon injections indogs. Intraocular pressure (IOP) and eye surface hyperemia were followedfor a month after injection. It was observed that a concentratedmicrosphere formulation provided longer IOP lowering effects.

Latanoprost containing microspheres injected were formulated with andwithout a HA (Captique or J18, both being partially cross-linked HAs).

The methods for injecting the dogs was as follows:

-   1. Baseline examination procedures were as follows for Beagle dogs.    Both eyes prior to application of test material had:    -   a. Recorded gross ocular features    -   b. External photographs were taken    -   c. IOP was measured    -   d. Pupil diameter (mm) was measured.-   2. Sedation was performed using an IV injection of Propofol and a SQ    injection atropine as a pre-anesthetic.-   3. After sedation, proparacaine and betadine ophthalmic solution    were applied.-   4. The injection was given through a 25 G gauge needle into the    superior temporal sub-tenon space.-   5. Ocuflox ophthalmic solution was applied BID for two days.-   6. Follow-up examinations was performed using the procedures in step    1.-   7. Follow-up schedule: Day 1, Day 2, Week 1, Week 2, Week 3, and    Week 4.

Results: FIG. 3 shows the concentration of the latanoprost containingmicrospheres used with and without hyaluronic acid (Captique). The useof a more concentrated suspension of latanoprost containing microspheres(i.e. the 50 ul formulation) and the use of the latanoprost containingmicrospheres in Captique permitted localization of the latanoprostcontaining microspheres in the episclera and also prevented excessivedispersion of the microspheres in the sub-Tenon's space. Thus we foundthat the 100 ul (less concentrated formulation) microsphere injectionwould readily disperse 180 degrees around the eye. Surprisingly, asshown by FIG. 3 some of the formulations reduced the IOP more thanXalatan® eye drops in the same dogs. IOP reduction by Xalatan® eye dropis shown by the “Max Topical Decrease” data point in FIG. 3. In FIG. 3the “50 ul” formulation consisted of 20 weight % latanoprost containingmicrospheres (total of 1240 ug latanoprost in the microspheres presentin the 50 ul suspension) and 80 weight % liquid vehicle (2%carboxymethylcellulose with 0.1% tween 80 in saline). The “100 ul”formulation consisted of 10 weight % latanoprost containing microspheres(total of 1240 ug latanoprost in the microspheres present in the 100 ulsuspension) and 90 weight % of the same liquid vehicle (2% CMC, 0.1%tween 80 in saline). Finally in FIG. 3 the “100 ul w/Captique”formulation consisted of 10 weight % latanoprost containing microspheres(total of 1240 ug latanoprost in the microspheres present in the 100 ulsuspension) and 90 weight % Captique gel (HA).

The results of using a higher concentration of latanoprost containingmicrospheres and also the use of an HA with the latanoprost containingmicrospheres was found to lower the IOP more efficiently and alsoprovided less conjunctival hyperemia. This may be the result because ifdrug dispersion is reduced, then there is less conjunctiva that hasexposure to the microspheres and thereby less clock hours of conjunctivathat show hyperemia.

An “anterior” intraocular location for the purpose of this Example meansa sub-Tenon, subchoroidal, suprachoroidal, intrascleral, episcleral, andthe like intraocular location which is located no more than about 10 mm(and preferably no more than about 8 mm and more preferably less thanabout 5-6 mm) along the curvature of the surface of the eye from thecorneal limbus. On the other hand a “posterior” intraocular location forthe purpose of this Example means into a location behind the equator ofthe eye, that is a location at least about 8-9 mm or more away from thelimbus. We compared injection of latanoprost containing microspheres inthe “anterior” vs “posterior” (as respectively defined above in thisExample) sub-Tenon space of the eye. We determined that an anteriorinjection location is preferred because that placed the microspheres inthe juxtascleral area adjacent to the ciliary body at what we call “theanterior portal”. Without wishing to be bound by theory we can proposean explanation as to why anterior sub-tenon injection of latanoprostcontaining microspheres can provide a greater (more IOP lowering) and/ora longer lasting IOP lowering effect. Thus, as shown by FIG. 4 aqueoushumor (AH) production occurs via ultrafiltration at the ciliary body(blue in FIG. 4 represents the ultrafiltrate fluid). The pathway of thefluid produced at the ciliary body is illustrated by the arrows, someenter the posterior chamber (area behind the iris), and then the fluidpercolates through the pupil and eventually into the trabecularmeshwork. Other fluid goes into the vitreous humor and the net fluidmovement is outward due to 1)hydrostatic Pressure and 2) osmoticpressure gradients in areas posterior to pars plicata region. Therefore,fluid movement in the vitreous posterior to the pars plicata region iscounter-directional to inward diffusion drug placed in the sub-Tenons(episcleral) space. FIG. 5 shows that drug (white color) such aslatanoprost that is diffusing is actually facilitated when diffusingfrom the anterior sub-Tenon area to the anterior chamber since the netfluid movement is inward in this area. More posterior, the net fluidmovement is outward and no drug (i.e. latanoprost) is shown diffusinginto the vitreous. Hence we determined that a facilitated pathway intothe eye exists and we cal it the “anterior portal pathway”.

To test the hypothesis of the anterior portal, and therefore theexistence of a preferred sub-tenon injection location (at the anteriorsub-Tenon so as to access the area to access the anterior pathwayportal), we injected latanoprost containing microspheres both sub-tenonanteriorly or posteriorly. Significantly, the posterior injection showedno IOP reduction. The anterior sub-tenon injection of latanoprostcontaining microspheres with or without a high viscosity hyaluronic aciddid show IOP reduction. Importantly, the microsphere-J18 formulationmaintained intraocular residency of the microspheres longer than didneat (in saline, no HA) injections of the microspheres.

We found that in vivo (sub-tenon in dogs) administration of latanoprostcontaining microspheres formulated with a high viscosity hyaluronic acidprovided a greater decrease of baseline IOP than did the samelatanoprost containing microspheres not formulated with a high viscosityhyaluronic acid. See eg FIG. 3. Captique (available from Allergan,Irvine, Calif. or from Genzyme Corp, Cambridge, Mass.) is a cross linkedhyaluron (hyaluronic acid) with an average molecular weight of about 2million Daltons. Similar results (greater change from IOP baseline uponsub-tenon administration in dogs) were obtained when the microspheresMP5 (total of 1242 micro grams of latanoprost contained by the sample ofMP5 used) were formulated with 50 micro liters of another high viscosityhyaluronic acid J18 (J18 is Juvederm 18, available from Allergan,Irvine, Calif. J18 is a cross linked (less than 6% cross linked)hyaluronic acid (18 mg in 1 ml of phosphate butter pH 7.2) with anaverage molecular weight of about 2 million Daltons, and a G′ value ofabout 60.

It was surprizing and unexpected to observe that latanoprost containingmicrospheres upon intraocular administration provide a greater decreasefrom baseline IOP when formulated with a high viscosity hyaluronic acid.It can be inferred therefore that in some way the presence of a highviscosity hyaluronic acid in the formulation (as a carrier for thelatanoprost containing microspheres) potentates or increases the IOPlowering effect of the latanoprost released from the microspheres.Additionally, ophthalmologists are adverse to injecting a bioerodiblepolymer drug delivery device (such as a composition within the scope ofthe present invention into an anterior sub-tenon because of the widelyheld belief they have that any polymer remnant will remain visible longafter injection, thereby providing a cosmetic deterrent to treatment bysuch an administration route. Contrary to this widely held belief wedetermined that anterior sub-tenon injection of latanoprost containingmicrospheres does not result at any time in a visible remnant.

The benefits of using microsphere in gel (high viscosity hyaluronicacid) formulation include that ease of the injection was significantlyimproved when comparing with suspension (no HA) formulation (no syringeclogging upon injection); storage stability (no microsphere settlingupon storage of the formulation), and formation of a concise andmaintained depot at the site of intraocular injection.

Example 3 Subconjunctivally Injected Latanoprost Microspheres to TreatGlaucoma

A 56 year old male suffering from glaucoma in both eyes receives asubconjunctival injection of PLGA microspheres containing latanoprost(as set forth in Example 2) made by the method of Example 1 within theanterior sub-tenon space of each eye. The microspheres contain 10 weight% latanoprost, and 20 mg of microspheres are used for each injection. Inabout two days, the patient reports a substantial relief in ocularcomfort. Determination of intraocular pressure (IOP) reveals that theIOP has decreased in each eye, the average intraocular pressure measuredat 8:00 AM has decreased from 28 mm Hg to 14.3 mm Hg. The patient ismonitored every other day for about 1 month. Intraocular pressure levelsremain below 15 mm Hg for this period of time, and the patient reportsreduced ocular discomfort. Little or no ocular hyperemia is observed andno iris color or pigmentation change is observed. Furthermore, themicrospheres nor any portion thereof cannot be seen at any time afterinjection when the patient is observed facing the observing at adistance of 3 feet or more with normal eye activity, without the eyelidsbeing depressed or the conjunctiva exposed for examination. In otherwords there was no cosmetic effect on any aspect of the patient'sappearance from the sub-tenon injection of the latanoprost containingmicrospheres.

Example 4 Method for Making Latanoprost Implants

Latanoprost is a prostaglandin F_(2α) analogue approved for treatment ofopen-angle glaucoma or ocular hypertension in patients who areintolerant of other intraocular pressure lowering medications orinsufficiently responsive (i.e. failed to achieve target IOP aftermultiple measurements over time) to another IOP-lowering medication.Latanoprost may be used alone or in combination with other antiglaucomaagents. Latanoprost is an isopropyl ester prodrug. It is hydrolyzed byesterases in the cornea to latanoprost acid, which is biologicallyactive. The elimination of latanoprost acid from plasma is rapid(half-life 17 minutes) after either ophthalmic or intravenousadministration. Thus, there is a need for a latanoprost sustainedrelease polymer implant, which can be uses as an effective treatment forlong-term reduction of intraocular pressure associated with glaucoma orother ocular diseases. In this Example we made sustained releasepolymeric implants containing therapeutically effective amounts oflatanoprost.

Latanoprost (molecular weight 432.58 and approximate pH 6.7) is acolorless to slightly yellow oil that is very soluble in acetonitrileand freely soluble in acetone, ethanol, ethyl acetate, isopropanol,methanol and octanol. It is practically insoluble in water.

Implants were made by hot-melt extrusion to contain 30 wt % latanoprost,40-60 wt % of a biodegradable poly (D,L-lactide-co-glycolide) polymer(Resomer® RG752s)(PLGA), 0-20% of a biodegradable poly (D,L-lactide)polymer (Resomer® R202s)(PLA), and 10% PEG-3350. The formulations and invitro drug release profile for the two implants made are summarized inTable 1 and FIG. 6, respectively.

TABLE 1 Latanoprost containing Implants Weight % Resomer ResomerFormulation No. Latanoprost RG752s R202s PEG-3350  8933-070 30 60 0 1028933-085 30 40 20 10

Manufacturing Method (Low Temperature Extrusion)

The latanoprost containing bioerodible polymer implants in this Examplewere made by hot-melt extrusion using a mechanically driven rammicroextruder but they can also be made by direct compression or solventcasting. The implants were rod-shaped, but they can be made into anygeometric shape by changing the extrusion or compression die. Polymers(the (resomers) were used as received from Boehringer Ingelheim andlatanoprost was used as received from Daiichi Fine Chemical Co., Ltd.

The samples were initially mixed (including the resomer powder and the10 weight % PEG) using a spatula in a weigh-boat for 15 minutes. Thesamples were then transferred into a stainless steel containercontaining two ¼″ stainless steel ball and mixing continued using aTurbula mixer for two separate 15 minute cycles. The powder blend wasmixed by hand using a spatula between each cycle and after the finalcycle. The blended material was then compacted into an extruder barreland the extruder barrel was placed into the heated well (between 50 and55 degrees C.) of the piston extruder and extruded using 500 pm nozzleand a speed setting number of 0.0025.

We developed this low temperature extrusion method because latanoprostis an oil at room temperature and begins to lose biological activity ifheaded above about 56° C. A low temperature extrusion is possible due touse of about 5-10 weight % PEG as a co-solvent for the resomers. Othersuitable PEGs which can be used in this method are PEG 5000 and PEG7000. Up to about PEG10,000 is suitable. Besides a PEG other suitableco-solvents include 5-10 weight % cholesterol and polyvinylprovidine.See eg U.S. patent application Ser. No. 11/612,928, filed Dec. 19, 2006.The method disclosed herein permitted extrusion at a temperature evenlower than that disclosed in application Ser. No. 11/612,928. Preferablythe resomers used have a relatively low molecular weight so as to formimplants which homogenously incorporate biologically active latanoprost,and so that the implant do not have significant burst release of drugupon intraocular administration. Thus the RG752s resomer has an inherentviscosity of from 0.16 to 0.024 dl/g and R202s resomer has an inherentviscosity of 0.2 dl/g and these resomers have average molecular weightsof about 11,200 and 6,500, respectively.

The extruded filaments were cut into one milligram implant(approximately 3 mm long), and (for in vitro release study) placed intoa 10 ml vial containing 0.01M phosphate buffered saline (pH 7.4), andthen transferred into a shaking water bath set at 37° C. and 50 rpm. Atvarious time points, the solution was removed and analyzed by HPLC todetermine the amount of latanoprost released by the implants. Theremoved solution was replaced with fresh phosphate buffered salinesolution. The release profiles observed are shown by FIG. 6.

Example 5 Subconjunctivally Injected Latanoprost Implant to TreatGlaucoma

A 64 year old female suffering from glaucoma in both eyes receivessubconjunctival administration of a PLGA/PLA implant containinglatanoprost made by the method of Example 4 within the anteriorsub-tenon space of each eye. The implant contains 30 weight %latanoprost. In about two days, the patient reports a substantial reliefin ocular comfort. Determination of intraocular pressure (IOP) revealsthat the IOP has decreased in each eye, the average intraocular pressuremeasured at 8:00 AM has decreased from 28 mm Hg to 14.3 mm Hg. Thepatient is monitored every other day for about 1 month. Intraocularpressure levels remain below 15 mm Hg for this period of time. Little orno ocular hyperemia is observed and no iris color changes is observed.

All references, articles, publications and patents and patentapplications cited herein are incorporated by reference in theirentireties.

While this invention has been described with respect to various specificexamples and embodiments, it is to be understood that the invention isnot limited thereto and that it can be variously practiced within thescope of the following claims.

We claim:
 1. A pharmaceutical composition for intraocular use to treatan ocular condition, the composition comprising: (a) a plurality ofmicrospheres made of a bioerodible polymer, and; (a) a therapeutic agentselected from the group consisting of latanoprost, bimatoprost andtravoprost and their salts, esters and derivatives, contained by themicrospheres.
 2. The composition of claim 1, wherein the microspherescomprise from about 1% to about 99% by weight of the polymer.
 3. Thecomposition of claim 2, wherein the polymer is a PLGA.
 4. Thecomposition of claim 2 wherein the microspheres have an average greatestdimension in a range of from about 5 microns to about 1 mm.
 5. Thecomposition of claim 4 wherein the microspheres has a mean diameterbetween about 15 microns and about 55 microns.
 6. The composition ofclaim 5 wherein the therapeutic agent comprises from about 0.1% to about90% by weight of the microspheres.
 7. The composition of claim 6 whereinthe microspheres comprise between about 8 to 15 weight % latanoprost. 8.The composition of claim 1 further comprising a high viscosityhyaluronic acid.
 9. The composition of claim 1 wherein the ocularcondition is glaucoma.
 10. A pharmaceutical composition for intraocularuse to treat glaucoma, the composition comprising: (a) a plurality ofmicrospheres made from a PLGA; (b) latanoprost contained by themicrospheres, and; (c) a high viscosity hyaluronic acid.
 11. Apharmaceutical composition for intraocular use to treat glaucoma, thecomposition comprising: (a) a sustained release implant made from a PLGApolymer, a PLA polymer, and a PEG co-solvent, and; (b) latanoprostcontained by the implant, wherein the implant comprises about 30 weightpercent latanoprost and the implant can release the latanoprost over aperiod of time of at least 70 days.